2 Mechanisms of DNA Methylation
Page 1: Overview of DNA Methylation
Assigned Readings:
Kadonaga. Perspectives on the RNA Polymerase II Core Promoter. Wiley Interdiscip. Rev. Dev. Biol. 2012
Tollefsbol, "Handbook of Epigenetics: Chapter 2"
Ooi et al., DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA. Nature 2007
Page 2: Controlling Transcription
Promoter Types:
Not all promoters are the same, including TATA, Inr, DPE, BRE.
Transcription Factor Influence:
Activators and repressors can modify transcription processes.
Chromatin Structure:
Will be discussed in the next lecture (Chapter 3).
Post-Translational Modifications (PTMs):
Observations on PTMs of DNA play a critical role.
Page 3: Promoter Understanding
TATA-Dependent Promoters:
Majority of understanding derived from TATA-dependent scenarios (10-20% of all promoters).
About one-third of promoters do not possess known motifs.
Inr (Initiator) Sequence:
Most prevalent is YYANWYY (A+1) in humans, regardless of predominant mRNA.
TFIID interacts; TATA box is bound by TBP at -30 relative to Inr.
Two BREs are uncommon; both can exert positive and negative effects on transcription.
Downstream core promoter elements are present in TATA-less scenarios (located +18 to +33).
TAF6 and TAF9 are also involved.
Reference: Kadonaga, 2013.
Page 4: Additional Promoter Details
TCT Motif:
Found in polypyrimidine initiators as YC+1TYTYY in humans, linked to translation genes.
Transcription Pausing:
Noted pausing 20-50 nt downstream from the start site, may utilize DPE/TAFs.
CpG Islands:
Represent 50% of known promoters and typically remain unmethylated.
Reference: Kadonaga, 2013.
Page 5: Focused vs. Dispersed Transcription Models
Models of Transcription:
Focused Transcription Model - Initiating transcription at a precise location.
Dispersed Transcription Model - Multiple weak start sites allow for resilience to DNA changes.
Pol II and TFIID Relationship:
Interaction between Pol II, TFIID and specific promoter-binding factors, e.g., Sp1.
Reference: Kadonaga, 2013.
Page 6: Focused vs. Dispersed Transcription - Practical Implications
Gene Control:
It is practical to control genes from specific start points.
Genes using multiple weak start sites display enhanced stability to minor DNA structural changes.
Page 7: Impact of Promoter Composition
Case Study: p53 Activation:
p21 Transcriptional Activation:
Fast activation, efficient PIC assembly, contains TATA box, promotes cell cycle arrest.
FAS Transcriptional Activation:
Slow activation, inefficient PIC assembly, TATA-less promoter that promotes apoptosis.
Page 8: Transition to Next Lecture
Lecture Part II:
Title: LIBERTY UNIVERSITY
Page 9: Summary of Promoter Control Features
Key Focus Areas:
Promoter diversity includes TATA, Inr, DPE, and BRE structures.
The role of transcription factors on modulation.
Overview of chromatin structure to be further elaborated in the next chapter.
Importance of PTMs on DNA.
Page 10: Methylation Overview
DNMT Types:
DNMT3 versus DNMT1
DNMT3L Role:
Protein Patterns:
Integration into heritable marks.
Mechanisms of Methylation:
Demethylation Process:
Focus on roles of DNA Methyltransferases (DNMTs).
Page 11: DNMT Variants and Functions
DNMT Families:
DNMT1, DNMT2, DNMT3a, DNMT3b, DNMT3L;
DNMT2 associated with RNA methylation playing a role in tRNA stability.
Page 12: DNMT Functionalities
Maintenance vs. De Novo Enzymes:
DNMT1 acts as maintenance enzyme for DNA methylation, while DNMT3 is involved in de novo methylation processes.
DNMT3L considered a 'dead' enzyme.
Page 13: Structural Features of DNMTs
DNMT1 Specifics:
Contains replication domains aiding in interaction with PCNA.
DNMT3L Characteristics:
Lacks PWWP domain for non-specific DNA binding and conserved catalytic sequences.
Page 14: Mechanistic Overview of DNMTs
De Novo vs. Maintenance Activity:
DNMT1 methylates hemi-methylated substrates, ensuring replication and maintenance process.
Focus on unmodified DNA and roles across methylation states.
Reference: Tollefsbol, "Handbook of Epigenetics: Chapter 2"
Page 15: Observational Insights
Stable CpG Patterns:
Hypothesis emerging from observations indicates stable CpG patterns may guide non-CpG DNA methylation. (Grandjean, 2007)
Page 16: Dnmt3L Phenotypic Observations
Dnmt3L Knock-Out Effects:
Similar phenotype between Dnmt3L and Dnmt3a knockouts;
Dnmt3L enhances de novo activity of the methylation complex and co-localizes with Dnmt3 enzymes.
Page 17: Interaction Details of Dnmt3L
3L and 3A Interaction:
Alteration of interfaces detrimental to the activity of the complex.
Dnmt3A dimer increases overall DNA binding efficiency, also interacts with H3 tails in nucleosomes.
Page 18: Methylation Efficiency of Dnmt3A
Binding and Methylation Mechanism:
Dnmt3A capable of methylating CpGs in close proximity (8-10 bp apart) in one binding event.
Page 19: Linking Protein Patterns to Inheritable Marks
Interconnections:
DNA methylation closely tied to histone modifications;
Dnmt3L interacts with both Dnmt3 enzymes and histones.
Epigenetic Correlation:
Indirect and direct correlations established between methylation patterns and histone modifications.
Page 20: Correlation Between Methylation and Histone Modifications
Conditional Relationships:
Methylation levels of histones correlate with local DNA methylation; essential histone modifying enzymes implicated in methylation changes across species.
Page 21: Reiteration of Key Topics
DNMT3 versus DNMT1.
DNMT3L.
Translating protein patterns into inheritable marks.
Methylation mechanisms.
Demethylation processes.
Page 22: Mechanisms of DNA Methylation
Key Players UHRF1 and PCNA:
UHRF1 targets DNMT1 to hemimethylated DNA for maintenance.
Mechanism Description:
Newly replicated DNA is only methylated on the 'old' strand, ensuring CpG patterns are maintained across generations.
Page 23: Base Flipping Mechanism
Mechanistic Insights of UHRF1:
Describes the base flipping mechanism where UHRF1 makes interactions with unmethylated cytosines leading to potential methylation.
Page 24: Understanding Demethylation Processes
Roles of MBD4:
Contains both methyl-binding domain and glycosylase activity.
DNMT1 Passive Loss:
Mechanisms involving enzymatic and chemical demethylation through varying processes including DNA repair.
Page 25: Types of Demethylation
Outcomes of Demethylation:
Deamination of methylcytosine yields thymine; illustrating transformation pathways from cytosine through enzymatic changes.
Page 26: Hydroxymethylation Understanding
Pathways of Cytosine Modifications:
Cytosine can undergo hydroxymethylation, often debated whether 5hmC is an end product or an intermediate substance in gene regulation.
Page 27: Introduction to Lecture Part III
Transition Notice:
Commencement of Lecture Part III at LIBERTY UNIVERSITY.
Page 28: Paper Discussion Guidelines
Critical Questions for Figures:
What question is being addressed and why?
What methodology is applied?
Key findings reported?
Notable experimental controls?
Additional questions raised by findings?
Page 29: Ooi et al. Figure 1 Analysis
Figure detailing interactions with DNMT3 variants and associated functionalities.
Page 30: Ooi et al. Figure 2 Analysis
Quantitative measurements of associated modifications (H3K4Me) in response to DNMT3L concentrations.
Page 31: Ooi et al. Figure 3 Analysis
Experimental responses of mutant DNMT3L variants underpinning binding efficiency and impacts on methylation outcomes.